The Na4+/Ca2+ exchanger is believed to play important roles in Ca2+ handling of excitable cells but its precise physiological functions have been difficult to determine due to the lack of specific inhibitors. The major goal of this proposal is to elucidate by cDNA cloning the structure of Na+/Ca2+ exchangers expressed in the brain and heart, and to raise, by a novel strategy, specific antibodies that can be used to define physiological roles of the exchanger in the cardiovascular system. To accomplish this goal, we will first complete cDNA cloning of the brain Na4+/Ca2+ exchanger by functional expression in Xenopus oocyte. Second, the full-length cDNA encoding the cardiac Na+/Ca2+ exchanger will be isolated by a combined use of low stringency hybridization and Xenopus oocyte expression system. Third, a series of monoclonal antibodies directed against outward-facing domains of the exchanger will be raised by a novel strategy. This involves hyperimmunizing mice with syngenic cells which overexpress the heterologous cardiac exchanger in its native, membrane-inserted conformation. Fourth, these monoclonal antibodies will be used to map the membrane topology of the exchanger in the lambda-gt11 expression-epitope mapping system. Fifth, we will screen for a monoclonal antibody that acts as a specific inhibitor of the ion transport function of the exchanger. If such a "blocking antibody" cannot be raised, we will develop a system to specifically inhibit the expression of the Na+/Ca2+ exchanger in cultured cardiocytes by an anti-sense oligonucleotide or by infecting cardiocytes with a non-cytopathic adenovirus vector that allows an efficient production of the anti-sense RNA. Finally, we will use the blocking antibody (or the anti-sense approach) as a pharmacological tool to define the roles of the Na+/Ca2+ exchanger in excitation-contraction coupling, digitalis action, arrhythmogenesis and Ca2+ overload during reperfusion injury. The "reverse pharmacology" approach we propose here may be applicable to the study of other clinically important membrane transport systems for which no specific ligands are currently available.